Inhibition of murine AIDS by pro-glutathione (GSH) molecules.

Antioxidant molecules can be used both to replenish the depletion of reduced glutathione (GSH) occurring during HIV infection, and to inhibit HIV replication. The purpose of this work was to assess the efficacy of two pro-GSH molecules able to cross the cell membrane more easily than GSH. We used an experimental animal model consisting of C57BL/6 mice infected with the LP-BM5 viral complex; the treatments were based on the intramuscular administration of I-152, a pro-drug of N-acetylcysteine and S-acetyl-beta-mercaptoethylamine, and S-acetylglutathione, an acetylated GSH derivative. The results show that I-152, at a concentration of 10.7 times lower than GSH, caused a reduction in lymph node and spleen weights of about 55% when compared to infected animals and an inhibition of about 66% in spleen and lymph node virus content. S-acetylglutathione, at half the concentration of GSH, caused a reduction in lymph node weight of about 17% and in spleen and lymph node virus content of about 70% and 30%, respectively. These results show that the administration of pro-GSH molecules may favorably substitute for the use of GSH as such.

Antiviral and immunomodulatory properties of new pro-glutathione (GSH) molecules.

Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro-GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.

[Oxidative metabolism of HIV-infected macrophages: the role of glutathione and a pharmacologic approach]. / Métabolisme oxydatif dans les macrophages infectés par le VIH: rôle du glutathion et approche pharmacologique.

Oxidative stress and glutathione deficiency seem to play a major role in the pathogenesis of HIV infection, as suggested by the increased survival of HIV-infected patients treated with N-acetylcysteine, a prodrug of glutathione. However, beneficial effects of GSH-replenishing drugs are restricted in vivo by the high concentrations needed to obtain biological effects and their low bioavailability. In this study, we evaluated the antiretroviral and antioxidant activities of new more lipophilic GSH-replenishing molecules, in macrophages infected in vitro with HIV-1. In these experimental conditions, a prodrug of N-acetylcystéine and beta-mercaptoethylamine, I-152 demonstrated a potent anti-HIV activity, increased intracellular GSH level, and decreased TNF-alpha production. Altogether, these results suggest that I-152 could be beneficial as adjuvant therapy of antiretrovirals in HIV-infected patients, especially in those with damages to the central nervous system or with mitochondrial damages associated with highly active antiretroviral therapy.

NAC/MEA conjugate: a new potent antioxidant which increases the GSH level in various cell lines.

I-152 is a prodrug of NAC and MEA with potent pro-GSH effects in human macrophages, astrocytes and lymphocytes. This molecule could be of interest in HIV infection in respect to its antioxidant and anti-HIV activities, but also in other diseases to counteract oxidative stress, that is, inflammation, cardiovascular diseases, and neurodegenerative diseases.

Synthesis and in vitro anti-HIV activity in human monocyte-derived macrophages of 2-oxothiazolidine-4(R)-carboxylic acid derivatives.

Oxidative stress and glutathione (GSH) deficit may play an important role in HIV infection pathogenesis, and oral administration of GSH-replenishing drugs such as N-acetylcysteine (NAC) and 2-oxothiazolidine-4(R)-carboxylic acid (OTC) may be associated with an increased survival rate of HIV-infected patients. Nevertheless, beneficial effects of these molecules are restricted in vivo by the high concentrations that are necessary to obtain biological effects, rapid extracellular metabolization, and low availability and plasma concentrations. We synthesized OTC derivatives that are more lipophilic than OTC and theoretically able to overcome these limitations and to generate, in addition to cysteine, other substrates of the gamma-glutamyl cycle. Their antiviral effects were investigated in human HIV-1/Ba-L-infected monocyte-derived macrophages. In our experimental conditions, OTC exhibited anti-HIV-1 effects and little cytotoxicity at high doses. None of the nine tested derivatives showed higher cytotoxicity than OTC, nor anti-HIV-1/Ba-L activity.

Metabolism of a new radioprotector; S-acetyl-N-glycyl cysteamine. II. Main tissue metabolites in mice bearing EMT6 tumours.

1. The major tissue metabolites of the radioprotector S-acetyl-N-glycl cysteamine (I) labelled with 14C on the cysteamine group, were quantified and identified in normal tissues and EMT6 tumours implanted in mice, by chromatographic comparison with authentic reference compounds. 2. In all tissues the radioprotector undergoes rapid deacetylation and hydrolysis leading to the formation of cysteamine, which is the main metabolite involved in radioprotection. A major part of this metabolite is reversibly inactivated by binding to endogenous SH. 3. The differential radioprotection of healthy tissues versus EMT6 tumour is explained both by a lower uptake of radioprotector, and a weaker deacetylation and hydrolysis rate, in the tumour cells.

Synthesis and radioprotective activity of dipeptide cysteamine and cystamine derivatives.

Some N-(dipeptidyl)-S-acetylcysteamine and N,N'-(dipeptidyl)cystamine salt derivatives were synthesized and evaluated as candidate radioprotector agents. Toxicity and radioprotective activity as the dose reduction factor (DRF) were determined in vivo on mice and compared to N-glycyl-S-acetylcysteamine trifluoroacetate. One of the most interesting compounds of this series was N-glycylglycyl-S-acetylcysteamine trifluoroacetate (8).

Metabolism of a new radioprotector; S-acetyl-N-glycyl cysteamine. I. Absorption, distribution and excretion metabolites in mice bearing EMT6 tumours.

1. The disposition of S-acetyl-N-glycyl cysteamine (I) labelled with 14C on the cysteamine group (label 1), the glycyl group (label 2) and the acetyl group (label 3) has been studied in mice bearing EMT6 tumours. 2. Label 1 was mainly excreted in urine (63.1% dose in 24 h). Label 2 elimination was both in urine (36.0% dose in 24 h) and in expired air as 14CO2 (12.1% dose in 24 h). Label 3 was essentially eliminated in expired air as 14CO2 (55.4% dose in 24 h). 3. Tissue distribution studies of label 1 and label 2 showed that concentrations in tissues were higher than blood concentration as early as 10 min after administration. Whichever label was used, only little radio-activity was found in EMT6 tumour and brain. 4. Analysis of the urinary elimination products showed the presence of unchanged I and of cystamine, N-acetylcystamine, N-acetyl-S-methyl cysteamine sulphoxide and taurine. I is a prodrug of cysteamine which is released after deacetylation and hydrolysis of the amide bond. A metabolic pathway is proposed for this new radioprotective agent.

Synthesis and radioprotective activity of new cysteamine and cystamine derivatives.

A variety of N-(aminoalkanoyl)-S-acylcysteamine and N,N'-bis(aminoalkanoyl)cystamine salt derivatives were synthesized. Toxicity and radioprotective activity (as the dose reduction factor DRF) were determined in vivo on mice and compared to WR 2721 and S-acetylcysteamine hydrochloride. One of the most interesting compounds of this series was N-glycyl-S-acetylcysteamine trifluoroacetate (16, I 102). Structure-activity relationships are discussed.

Radiobiological evaluation of a newly synthesized cysteamine derivative.

A new cysteamine-based compound, I109 (N-glycylglycyl-S acetylcysteamine trifluoroacetate) was tested on both normal tissues and tumors to evaluate its radioprotective potential. I109, which is three times less toxic than WR-2721, was injected at a dose approximately equal to half the LD50(30 days). The Protection Factor (PF = gamma ray dose ratio) after whole body irradiation was 1.3-1.4 for intestinal death and 1.4-1.5 for hemopoietic death when intervals of 40 or 20 min elapsed between injection and the end of irradiation. A crypt cell assay was done for both I109 and WR-2721; PFs were 1.1 and 1.4, respectively. I109 was then tested on five solid tumors. For each cell line (4 human, 1 murine) one dose of radiation was delivered. Surviving fraction ratios with and without drug ranged between 2.4 and 14.1 when an interval of 20 min elapsed between injection and the end of irradiation. The degree of radioprotection proved time dependent for EMT6 and HRT18; radioprotection afforded by WR-2721 on these tumors is either similar or greater than radioprotection afforded by I109 depending on the time interval between injection and irradiation.

Radioprotection of EMT6 tumor by a new class of radioprotectors based on a pseudo-peptide cysteamine combination.

Although WR-2721 preferentially protects normal tissues against irradiation, it seemed desirable to find other drugs presenting a lower toxicity and the same radioprotective properties. A new compound, I 102, was selected; it was characterized on one hand by a coupling between cysteamine and an amino-acid, and on the other hand by an acetyl-group, which protects the thiol function. The effects of WR-2721 and of I 102 were studied on EMT6 tumors grafted on BALB/c mice. Whatever the size of the tumor, the cell survival increased as a function of the time elapsed between the injection of I 102 and the end of the irradiation (TI). In contrast, the radioprotection afforded by WR-2721 was found to be independent of TI. The survival curves suggest that, like WR-2721, I 102 protects essentially oxygenated cells.

Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid. Pathways of aqueous decomposition and pharmacological characteristics of new anticancer disulfide-linked nitrosoureas.

We have examined the pharmacological characteristics of three dinitrosated isomers of N,N'-bis[N(2-chloroethyl)-N-carbamoyl]cystamine [CNCC-(D), 1C1G1325] differing in the relative positions of the nitroso substituents [CNCC-(C), (1,1' dinitroso); CNCC-(S), 3,3' dinitroso); and CNCC-(M), (1,3'-dinitroso)] and which were designed to be subject to preferential bioreductive activation in hypoxic tumors. The decomposition products of the isomers formed under physiological conditions [both in the absence and in the presence of dithiothreitol (DDT)] were identified and quantified. For example, CNCC-(S) in phosphate buffer, pH 7.0, and 37 degrees gave rise to 2-chloroethylisocyanate, bis(2-chloroethyl)urea and bis(2-hydroxyethyl)disulfide, whereas in the presence of DTT it afforded 2-chloroethylisocyanate, bis(2-chloroethyl)urea, bis(2-hydroxyethyl) disulfide, thiirane and 2-mercaptoethanol. Control aqueous decomposition profiles were performed with two known metabolites of CNCC, namely 3-(2-chloroethyl)-1-(2-thioethyl)-1-nitrosourea and 3-(2-chloroethyl)-1-(2-methylthioethyl)-1-nitrosourea. CNCC-(C) caused 20% interstrand cross-linking of lambda-DNA in 2 hr, whereas in the presence of DTT the extent of cross-linking increased to 38% in the same time period. In contrast, isomer (S) showed no detectable cross-linking in 7 hr. This thiol potentiation of cross-linking which is observed with other 2-chloroethylnitrosoureas is explained by nucleophilic attack at the carbonyl group and subsequent stereoelectronically controlled decomposition of the tetrahedral intermediate. The relative extents of carbamoylating activity of the CNCC isomers were obtained using a [14C]-lysine assay which showed (S) approximately equal to (M) greater than (C). Inhibition of glutathione reductase for both Walker 256 resistant (WR) and Walker 256 sensitive (WS) strains showed that isomer (S) inactivated the enzyme more effectively than isomer (C) in accord with the carbamoylating activity results. The higher carbamoylators (S) and (M) also showed greater effects on the intracellular thiol pools in both WR and WS cells indicative of sulfhydryl conjugation and efflux and/or inhibition of the GSH metabolic enzymes. In vitro cytotoxicity studies with human DU 145 prostatic carcinoma cells showed the isomer cytotoxicity was (M) greater than (C) greater than (S) over a 24-hr incubation period. The reduced cytotoxic potential of CNCC-(S) in both the Walker 256 cells and in the human prostatic carcinoma cells may be a function of an interaction between GSH and the drug thereby protecting other more critical nucleophilic targets within the nucleus.

Activated N-nitrosocarbamates for regioselective synthesis of N-nitrosoureas.

A practical and convenient method for synthesizing antitumor compounds, N-alkyl-N-nitrosoureas, regioselectively nitrosated on the nitrogen atom bearing the alkyl group is proposed. N-Alkyl-N-nitrosocarbamates are interesting intermediates in these syntheses and yield, by reaction with amino compounds, the regioselectively nitrosated N-alkyl-N-nitrosoureas. As an interesting example, N,N'-bis[(2-chloroethyl)nitrosocarbamoyl]cystamine, a new attractive oncostatic derivative, has been prepared. The cytotoxic activity of these various compounds were tested on L1210 leukemia.

Action of three nitrosoureas on human lymphocytes. Hypothesis of a specific effect on lymphocyte subpopulations.

RFCNU [(chloro-2-ethyl)-1-(ribofuranosylisopropylidene-2',3'-paranitrobenzoate-5')-3- nitrosourea], RPCNU [(chloro-2-ethyl)-1-(ribopyranosyltriacetate-2',3',4')-3-nitrosourea] and CCNU [(chloro-2-ethyl)-1-cyclohexyl-3-nitrosourea) were found cytotoxic in vitro for human blood lymphocytes from healthy donors at doses higher than 50 micrograms/ml. Blood lymphocytes from different individuals displayed different behaviors both in their sensitivity to nitrosoureas and in their proliferative response to mitogenic lectins. With pokeweed-mitogen-stimulated lymphocytes the main effect of all three drugs (10 micrograms/ml) was an increase of [3H]-thymidine uptake. This facilitation effect was found only in half of the experiments with phytohemagglutinin-stimulated lymphocytes, while the stimulation by concanavalin A was inhibited under the same conditions. The hypothesis that nitrosoureas might be selectively cytotoxic for a suppressor cell subpopulation might explain the observed increase of thymidine uptake.

Flow cytofluorometric analysis of the effects of new nitrosourea derivatives on proliferation of EMT 6 tumor cells "in vitro".

Examined by flow cytofluorometric analysis, the DNA distribution of EMT 6 tumor cells was highly perturbed after one hour of in vitro incubation with: RPCNU, RFCNU, chlorozotocin (CZT) or 185 (CNCC), four new nitrosourea derivatives. After the treatment with chlorozotocin (20 micrograms/ml) and CNCC (50 micrograms/ml), most of cells were in G2 + M phase and this accumulation lasted more than 48 hours without any restoration before 72 hours. RPCNU (20 micrograms/ml) and RFCNU (50 and 65 micrograms/ml) induced and accumulation of cells in G2 + M phase during 24 hours. The normal state was regained after 48 hours. These reduced rate of progression of the cells through S phase and the G2 block observed after exposure to the new compounds, should, in part, explain their antitumoral activity.